Although viral vectors may deliver a transgene to liver cells with high efficiency, they also induce serious inflammatory response which precludes the practice of repeated injection. Non-viral vectors, however, although unable to produce ultra-high levels of gene expression in the liver, have the advantage of not inducing any inflammatory reactions. Accumulative or additive biological responses which are often sufficient for therapeutic purposes are readily achievable through repeated of a non-viral vector. Dr. Human has recently developed an emulsion system resembling human chylomicron remands with hydrophobic DNA/cationic lipid complex solubilized in the interior oil phase of the emulsion. Intraportal injection of the vector into mice results in approximately 10% of liver cells (mostly hepatocytes) expressing the transgene. Although the transgene expression lasts only about one week, a second injection at day 7 fully restores expression to initial levels. This emulsion vector will serve as a prototype for the development of future vectors. The aims of this vector development project are as follows: (1) To develop intravenously injectable, liver-specific vectors by conjugating hepatocyte-specific ligands to the surface of the vector. Ligands to be conjugated to the vector are triantennial galactosl moiety linked to a lipid asialoglycoproteins, and the malaria CS proteins. Vectors to be used in the study will include the reconstituted chylomicron remnant which naturally which naturally home to hepatocytes and condensed particles. (2) To construct hepatocyte-specific expression plasmids with prolonged transgene expression activity.Hepatocyte specific promoters such as the albumin promoter will be used. The transgene will be flanked with the inverted terminal repeat sequence of adeno-associated virus to promote stability in the nucleus, resulting in prolonged gene expression. Inclusion of an intron and a Kozak sequence at the 5 end of the transgene coding sequence is expected to further enhance the transgene expression level. Human alpha-1 antitrypsin and factor IX genes will be used as a secretory gene product to monitor long-term transgene expression. (3) To study the mechanism and trafficking of liver specific vectors in primary murine hepatocytes. The investigator will use two methods. To track DNA alone, the investigator will use radio-labelled (125I) DNA incorporated into the vector coupled with a combination of light and electron microscope autoradiography. This will allow the endpoints of DNA delivery to be studied in a quantitative, high resolution fashion. To actually examined the process of DNA/vector uptake, the investigator will use two color fluorescence labeling of DNA and vector. This technique allows tracking of the vector and DNA into cell and nucleus. Detection and analysis will be performed using a combination of confocal and live cell video imaging and immunogold electron microscopy. Emphasis will be placed on the uncoating of DNA from lipids, escaping from the endosome and entry into the nucleus. These studies will hopefully lead to the development of a safe and effective gene therapy vector for treating human diseases such as alpha-1-antitrypsin deficiency, factor IX deficiency and other liver- related metabolic disorders.